Ciliary beating plays a critical role in host defense in the human airway, but we know little about the endogenous signals that control ciliary beat frequency, or how the signals are transduced from cell surface receptors to effector proteins in the cell. We hypothesize that physical stresses induce luminal nucleotide release to activate autocrine and paracrine pathways that modulate components of mucociliary clearance., including ciliary beating. Nucleotides, acting via P2Y2 receptors, and adenosine, acting via A2B receptors, stimulate ciliary beat frequency but the mechanisms involved are poorly understood. We will use functional and biochemical assays to study the polarity of nucleotide/nucleoside regulation of ciliary function in airway. We will determine whether G protein coupled receptors are present on ciliary membranes generate cAMP (A2B receptor) and Ca2+/IP3 (P2Y2 receptor) locally, or whether the receptors are present on apical membrane microvilli, requiring the diffusion of second messengers into the ciliary shaft. We will also study the distribution of IP3 receptors to determine whether IP3 generate by activation of P2Y2 receptors acts on Ca2+ stores in the cell body, in the ciliary, or in both compartments. P2Y2 receptors are present on apical and basolateral surfaces, but agonist increases ciliary only when applied to the luminal surface. We hypothesize that mitochondria impose a diffusion barrier critical for the compartmentalization of Ca++ signaling in airway cells, and will test this hypothesis in epithelial sheets and in isolated cells. We will also study the compartmentalization of protein kinase A, the final effector of the A2B receptor pathway, because our results indicate that protein kinase A is anchored in the cilia by association with a novel A-kinase anchoring protein, and that the anchored kinase preferentially phosphorylates a single ciliary target. We will study the functions of anchored PKA, the novel ciliary anchoring protein, and the major protein phosphorylated in isolated cilia. Finally, we will determine whether the pathways we identify are activated when airway epithelial cells are exposed to physical stresses typically seen by the respiratory epithelial, including flow, vibration, and shear. Together our studies will elucidate will elucidate mechanisms involved in modulating cilia function in human airway in response to luminal stimuli and may lead to novel therapeutic strategies to enhance clearance in airway disease.